No Protein Detected: Target Protein Expression Issues (Immunoprecipitation (No Protein Detected))

Question

Accepted Answer

No target protein signal detected in IP eluate. Input lysate may also show weak or absent target protein signal when analyzed separately. Common causes: 1) Target protein is not expressed in the cell type or tissue used for sample preparation 2) Target protein expression level is below detection threshold in the sample 3) Insufficient starting lysate amount used relative to low target protein abundance 4) Target protein expression is restricted to specific conditions, developmental stages, or cellular compartments not present in sample Solutions: 1) Verify target protein expression profile using databases (Human Protein Atlas, GTEx) to confirm expression in sample cell type 2) Analyze input lysate by Western blot before IP to confirm target protein presence and estimate abundance 3) Increase starting lysate amount 2-5 fold (e.g., from 500 µg to 1-2.5 mg total protein) to capture low-abundance targets 4) Pre-clear lysate with plain beads (no antibody) for 1 hour at 4°C before IP to reduce non-specific binding when using increased lysate amounts 5) Consider using cell stimulation, transfection, or enriched cellular fractions to increase target protein levels